This invention relates generally to immunoassays, and more particularly, relates to a semi-automated assay system to detect and confirm the presence of antibodies, which assay system is an improvement over currently available assays for detection and confirmation of the presence of antibodies.
The human immune system responds to infections by generating antibodies to various antigens. The number of antibodies and the amount of each different antibody produced depends on the infectious agent and the particular antigen(s) which initiate the antibody response.
Current commercially available screening tests available for determining infections are based on enzyme immunoassays (EIAs) to detect the presence of antibodies. Generally, such tests utilize a solid support such as a polystyrene bead or microtiter well coated with a partially purified lysate containing antigens of the infectious agent. A test sample is contacted with the coated solid support, and any antibody which is present in the test sample and specific to the antigen is captured on the solid support, thus forming an antigen-antibody complex. This antigen-antibody complex is contacted with a second antibody labelled with a signal-generating detectable conjugate to form an antigen-antibody-anti-antibody complex. The presence and the amount of antibody in the test sample is determined by detecting the signal produced by the conjugate complex. However, the partially purified lysates may contain impurities and disproportionate quantities of various antigens or may lack important antigenic polypeptides. These deficiencies detrimentally affect both the sensitivity and specificity of the assay. Furthermore, it is often difficult to prepare sufficient quantities of partially purified lysates for traditional confirmatory tests, and the lack of a desired antigen increases purification problems.
These screening tests also have known non-specific reactivities. See, for example, J. E. Menetove et al., Lancet: 1213 (Nov. 21, 1987); D. Barnes, Science 238: 884-885 (1987); A. Puckett et al., Lancet: 714 (Mar. 26, 1988). A majority of the non-specificity is attributable to immunoreactivities with normal human antigens that are present in these lysates of the infectious agent. The normal human antigens (cellular protein impurities) originate from the tissue culture cells or other culture media that are used to propagate the infectious agent, and they are the major constituents of partially purified lysates.
The major surface antigen of Human Immunodeficiency Virus (HIV), gp120, and the HIV transmembrane antigen gp41 are readily lost during viral purification. H. Gelderblood et al., Virology 156: 171-176 (1987). These HIV envelope antigens thus often are underrepresented in viral lysates.
Consequently, a confirmatory test is required to corroborate reactive results obtained with the currently available licensed screening tests for HIV. The protocol for performing such confirmatory tests typically involves repeating the screening test to ensure a reactive test sample, and then performing a Western blot (WB) test on these repeat reactive test samples to confirm the reactive screening result. Those test samples which also are reactive by the WB test are considered as confirmed positive for HIV exposure.
The typical Western blot test is described by Gordon et al., U.S. Pat. No. 4,452,901. This test initially involves separating the antigens of a viral lysate according to size by SDS-PAGE under reducing conditions. The invisible bands of antigens in the gel then are electrophoretically transferred (transblotted) intact onto a sheet of nitrocelluose. This sheet then is cut into strips perpendicular to the bands. The strips are allowed to react with individual test samples and the reactive antibodies are detected by EIAs analogous to the screening assay. The resultant color reaction yields a pattern of stained bands revealing those antigens for which the test sample is seropositive. The WB test therefore has an advantage over screening tests because the antigens in the viral lysate which are reactive with the individual antibodies can be visualized separately. Additionally, non-viral reactivities in the inter-band areas can be identified and disregarded.
Despite the recited advantages of the Western blot test over traditional screening assays, the WB test has its drawbacks. See, for example, G. Biberfeld et al., Lancet ii: 289-290 (1986); C. L. Van de Poel et al., Lancet ii: 752-754 (1986); and A. M. Courouce, Lancet ii: 921-922 (1986). Traditional immunoblot methods such as the WB test lack reproducibility because viral lysates may vary in antigenic concentration.
The WB test reportedly lacks sensitivity, particularly in regard to the HIV envelope (ENV) antigens. A. Saah et al., J. Clin. Micro. 259: 1605-1610 (1987). We have observed that this is partly due to the preferential loss of the major surface antigen gp120 during virus maturation and purification, and partly due to the fact that gp120 is reduction sensitive. Its antigenicity is especially reduced in WB tests that utilize reducing SDS-PAGE. Further, the serologically most important HIV transmembrane ENV antigen gp41 (M. G. Sarngadharan et al., Ann. Inst. Pasteur/Virol. 138: 133-136 [1987]) characteristically gives a diffuse band on Western blots due to variable glycosylation; this band is quite weak when compared to the band produced by the p24 antigen. Moreover, the gp41 reactive band is especially weak when compared with the blots which utilize recombinant p41 antigen.
The Western blot test also yields a number of non-specific reactivities. The non-specific reactivities yield bands that sometimes overlap the true antigen bands. For example, we have observed that p24-only, p17-only, atypical gp41, or p70-only reactivities are those most likely to be false reactivities.
Further, the WB test yields extra bands of reactivities that are partly due to partially cleaved or uncleaved viral precursor proteins which may appear between the main antigenic bands. These extra reactivities also may be due to aggregations or degradations of viral antigens. For example, an HIV gp120 band may be due to a trimeric aggregation of gp 41. S. Zolla-Pazner et al., New England J. Med. 320: 18-19 (1989). Both HIV gp41 and p24 are known to yield dimers, trimers, and tetramers despite reducing conditions during electrophoresis. Using monoclonal antibodies, we have observed that HIV gp120 can give rise to two other bands, gp80 and gp45. These two bands represent the N-terminal 2/3 and C-terminal 1/3 of gp120, respectively. They are most likely degradation products in viral lysates resulting from a specific cleavage of the gp 120.
Further problems with the WB test include minor bands of reactivity due to the presence of regulatory proteins such as nef, VPR, VPV, etc; the amount of time required for the test which usually involves an overnight incubation step; the manual nature of the test; the subjectiveness of the test results due to the visual readout; and the ability to detect only one strain of a particular virus per sample tested. Thus, the WB confirmatory test exhibits poor sensitivity, particularly in regard to the important HIV ENV markers, with an abundance of additional non-specific and specific bands and technical problems which make band identification difficult and turn-around time long.
The problems associated with using lysates of infectious agents in the WB test partially can be avoided by expressing desired antigenic polypeptides in various heterologous cell systems. These systems use recombinant techniques to insert the infectious agent genes of interest in cells that can produce quantities of these polypeptide products. This procedure often results in the production of the desired antigen in sufficient quantities, but typically is associated with the presence of new impurities and degraded antigenic fragments. Antigens produced by these methods also may form aggregates that must be minimized by using reducing agents or require the use of multiple gels to avoid both aggregation and degradation problems.
The use of several antigens in a single assay has been described. Lin et al., J. Virol. 59: 522-524 (1986) describes a dual antibody probing technique that permitted identification of Epstein-Barr virus and different herpes virus antigens in the same Western Blot test.
It also is possible to use multiple detection methods on a single Western blot test. Lee et al., J. Immuno. Methods 106: 27-30 (1988) describes a technique which uses sequentially applied sets of probing antibodies, enzyme-conjugated developing antibodies and enzyme substrates to detect two or more types of interferon on a single Western blot test. The same result can be achieved by simultaneously applying more than one type of probing antibody using a mixture of different enzyme-conjugated developing antibodies followed by successive applications of different substrates.
Gordon et al., European Patent Application Publication No. 0 063 810, published Mar. 11, 1982, describe immunoassay devices and kits comprising antigens or antibodies or both bound to a solid support. The use of the described solid supports makes possible a number of simultaneous antibody-antigen reactions in one operation. Gordon et al. describe applying single or successive doses of solutions of antigens or antibodies to the surface of the solid support using a pipet or syringe. In a preferred embodiment, the antigen is applied as a microdot formed by adding small volumes of an antigenic solution. The application however does not describe immunoassays using antigens purified from a complex mixture of proteins.
Lefkovits, WO 87/03965, published Jul. 2, 1987, describes a test strip for several simultaneous assays. The test strip is made from nitrocellulose impregnated with an antibody that is cut into strips and mounted on an inert backing. The soaking of a support sheet in a solution of antigenic peptide and then cutting the sheet into strips also is described, but the transferring of the antigen to the solid support electrophoretically is not taught in the publication.
It would be advantageous to provide an assay system which could be used as a confirmatory test in place of the Western blot or Western blot-based tests. Such an assay system would utilize highly purified monomeric antigens of infectious agents which are essentially free from impurities, aggregates or degraded fragments. This assay system therefore would have improved specificity compared with currently available screening and confirmatory tests. It also would be advantageous to provide an assay system which is semi-automatable with a non-subjective readout and therefore easier to evaluate than the WB test. Another advantage of such an assay system is the ability to use it as a research tool to better understand patterns of serological markers associated with viral infection, particularly in the case of Hepatitis C Virus, for which the virus has not been isolated. It additionally would be advantageous to provide a test which could differentiate HIV-1 and HIV-2 from each other by providing side-by-side comparative data, thus a test that is capable of detecting both HIV-1 and HIV-2 and also capable of distinguishing one type from the other. It further would be advantageous to provide an assay system which would allow for the simultaneous detection of a panel of multiple antigens or antibodies.